The field of RNA interference has attracted massive attention in recent years, as it can provide specific gene knockouts. Obviously, this is very important in basic research when studying genetic and biochemical pathways or the function of individual genes and gene products. In line with this, RNA interference has become a very important tool for target validation in the pharmaceutical industry. Moreover, substantial investments are made with the goal of developing RNA complexes capable of mediating RNA interference that can be used as drugs.
The attractiveness of RNAi for use in therapy lies in its sensitivity and sequence specificity. However, concerns have arisen concerning sequence specificity, e.g. because the wrong strand of the RNA complex may direct the response to the wrong target nucleic acids. Moreover, RNA complexes of a certain size induce a non-specific interferon dependent response, which is also undesirable.
Patent application US2003/0108923 describes RNA complexes capable of mediating RNAi comprising an antisense strand and a passenger strand, wherein the strands are 21-23 nucleotides in length. It is suggested that the RNA complexes are used for therapeutic applications.
Similarly, patent application US2005/0234007 describes RNA complexes capable of mediating RNAi comprising an antisense strand and a passenger strand, wherein the complex comprises 3′-overhangs. It is suggested that the RNA complexes are used for therapeutic applications.
WO2005/073378 describes RNAi complexes capable of mediating RNAi comprising an antisense strand and a passenger strand. The RNA complexes described in the specification comprise LNA residues and it is stated that incorporation of LNA residues near the 5′ end of one of the strands can control which strand is incorporated in the RISC complex, because the strand that forms the weakest base pair at its 5-end is incorporated into the RISC complex. Matranga et al. (2005, Cell Vol 123, pp 607-620), discloses that the maturation of the active RISC complex requires the cleavage of the passenger strand by Ago-2. The cleavage of the passenger strand occurs between nucleotides 9 and 10 during RISC assembly.
Leuschner et al., (EMBO Reports, published online 20 Jan. 2006) discloses RNAi-induced silencing complexes which have a discontinuous passenger strand. Leuschner et al also used 2′-O methyl ribose units at position 9 of the passenger strand (5′ to 3′). The RNAi complexes are tested in an in vitro cell extract experiment. The use of discontinuous passenger strands was found to result in efficient target RNA cleavage, as did RNAi complexes where the 2′-O methyl ribose unit was located at the passenger site cleavage site (9). However, when the 2′-O methyl ribose unit was located further upstream of the cleavage site there was a reduction in target RNA cleavage.
Neither Leuschner et al nor Matranga et al. disclose or suggest that RNAi-induced silencing complexes which have a discontinuous passenger strand are preferable RNAi complexes for use in therapy.
The use of synthetic siRNAs in vivo is currently hampered by lack of efficient means of siRNA delivery, low blostabilily in biological fluids and low specificity of action due to inherent gene off-target effects associated with the microRNA-like behaviour of all investigated siRNAs (Jackson, A. L et al., (2003) Nat Biotechnol, 21, 635-637; Birmingham, A et al., J. et al. (2006) Nat Methods, 3, 199-204; Jackson, A. L et al., (2006) Rna, 12, 1179-1187.). Several attempts to reduce off-target effects through chemical modification of synthetic siRNA have been made (Jackson, A. L et al., (2003) Nat Biotechnol, 21, 635-637; Birmingham, A et al., J. et al. (2006) Nat Methods, 3, 199-204; Jackson, A. L et al., (2006) RNA, 12, 1179-1187; Elmen, J et al. (2005) Nucleic Acids Res, 33, 439-447; Jackson, A. L et al. (2006) RNA). Since both sense- and antisense-strands can contribute to off-target effects (Jackson, A. L et al., (2003) Nat Biotechnol, 21, 635-637), minimizing sense strand incorporation into activated RISC should significantly increase targeting specificity and thereby reduce sense strand off-targeting. It is well established that the siRNA strand with the thermodynamically least stable 5′ end is preferentially utilized as antisense strand in activated RISC (Schwarz, D. S et al., (2003) Cell, 115, 199-208.).
Double stranded RNA complexes can mediate various modifications of target nucleic acids in the cell. In this process, the antisense strand of the complex acts as a guide, as the antisense strand can hybridise to target nucleic acids that have stretches of sequence complementarity to the antisense strand.
Before targeting a target nucleic acid, the antisense strand is often incorporated into an RNA guided protein complex (RGPC), which can act upon the target nucleic acid. One example of a RNA guided protein complex is the RNA Induced Silencing Complex (RISC). It is believed that other such RGPCs exist and that the RNA complexes of the present invention will also be of advantage, when used with these other RGPCs.
However, when used in vivo as a therapeutic agent or gene discovery tool, the silencing complex such as RISC is unable to distinguish which of the two strands of a siRNA silencing complex is the intended antisense strand, and which is the passenger or guide strand. The loading of a passenger strand into the silencing complex may well result in unintentional silencing of off-targets, i.e. unintentional targets which have sufficiently high complementarity to the passenger strand. The risk of such off-target events is therefore a major issue when considering the development of both therapeutic and gene discovery agents based upon siRNA complexes.
The selection of the strand for insertion into the RISC complex depends, in one aspect, upon the strength of the hydrogen bonding between the 5′ of each strand. By designing siRNAs to ensure the 5′ base of the passenger strand is a G or C and the 5′ base of the antisense strand is a A or T, it is possible to preferentially bias the selection of the antisense strand for incorporation into the RISC silencing complex. However this does not prevent loading of the passenger strand into the RISC silencing complex.
The incorporation of affinity enhancing nucleotide analogues at the 5′ end of the passenger strand can further reduce the proportion of passenger strand loading into the RISC silencing complex. Accordingly, selective thermodynamic stabilization of sense strand 5 ends by Incorporation of locked nucleic acids (LNA) has been shown to reduce unwarranted gene silencing by the sense-strand (Elmen, J et al. (2005) Nucleic Acids Res, 33, 439-447; Petersen, M. and Wengel, J. (2003) Trends Biotechnol, 21, 74-81.).
Incorporation of nucleotide analogues at positions 10 and 12 (from the 5′ end) of the passenger strand can prevent the RISC cleavage event as these residues are thought to align to the catalytic centre of the RISC complex. However, the incorporation of affinity enhancing nucleotide analogues reduces the efficacy of the modified siRNA complexes, possibly by increasing the resistance of the siRNA to the action of the RISC complex helicase. The incorporation of high loads of affinity enhancing nucleotide analogues has therefore been limited due to the negative effect such analogues have on silencing efficacy.
The introduction of dsRNA complexes into a mammalian cell can result in induction of the interferon response, which leads to cell death. Whilst it has previously been considered that the interferon effect has been limited to the presence of longer dsRNA molecules, such as those associated with viral infection and replication, it is now known that short siRNA like entities can also induce the interferon response. It has been suggested that the introduction of nucleotide analogues within the siRNA can be used to limit or even prevent the induction of the interference effect. Therefore, it has been considered desirable to introduce nucleotide analogues into siRNA like complexes for use in in vivo applications.
Incorporation of nuclease resistant nucleotide analogues is thought to be beneficial to protect the 3′ overlapping ends of smRNAs. As the 3′ overlaps do not contribute to the strength of the hybridisation between the sense and passenger strand these do not have a negative effect on the action of the RISC complex helicase.
There is therefore a critical problem which limits the success and efficacy of siRNA in in vivo applications such as therapeutic and gene discovery applications, how to prevent off target effects due to unintentional silencing caused by the passenger strand, whilst avoiding the undesirable inhibitor effects associated with the use of affinity enhancing nucleotide analogues.